NOX-ROS-TRPM2信号通路在肺高压发病过程中的作用

Pulmonary hypertension (PH) is a pathophysiological condition associated with a board spectrum of diseases of different pathological features and etiologic mechanisms. It is a severe disease characterized by increase in pulmonary vasomotor tone and progressive remodeling of the pulmonary vasculature, resulting in elevated pulmonary vascular resistance and leading to right heart dysfunction and death. Even though recent therapies can partially alleviate PH symptoms and lengthen patients' life expectancy, PH is still incurable and always worsens the prognosis of the underlying diseases. This clearly shows the need for a comprehensive understanding of the cellular and molecular mechanisms of PH in order to define new therapeutic strategies for combating this disease. Reactive oxygen species(ROS) play a pivotal role in the pathogenesis of many cardiovascular diseases. There is consistent evidence indicating that PH is associated with an increase in oxidative stress. Previous studies from several groups including ourselves showed that chronic hypoxia (CH)-induced PH is associated with enhanced superoxide and H2O2 production by NADPH oxidases (Nox) and upregulation of Nox4 in pulmonary arterial smooth muscle cells (PASMCs); deletion of Nox2 gene abolished chronic hypoxia induced PH. However, how ROS signals generated from Nox are translated into the pathological changes of PH is unclear. One possibility is through ROS-dependent Ca2+ pathways, because the enhanced pulmonary vasoreactivity, proliferation and migration of PASMCs, as well as many other processes of vascular remodeling in PH are Ca2+ dependent. We have characterized Ca2+ mobilization activated by ROS in PASMCs and discovered a major component of H2O2-induced Ca2+ influx, which is independent of voltage-gated Ca2+ channels, store-operated Ca2+ entry and receptor-operated Ca2+ entry. We have further identified in PASMCs the expression of transient potential receptor melastatin 2 (TRPM2), which is a non-selective Ca2+ permeating cation channel and is known to be activation by H2O2 and oxidative stress in other cell types. Based on these observations, we propose that Nox/ROS/TRPM2 is a major signaling pathway for the development of PH. To test this hypothesis, we will use a combination of biophysical techniques of electrophysiology and Ca2+ imaging, molecular techniques of gene knockdown and overexpression, and physiological techniques on three different models of PH of distinctively different etiologies to (1) determine the involvement specific Nox isoforms in the different forms of PH, (2) determine the activation mechanisms of TRPM2 in H2O2 (ROS)-induced Ca2+ response, and (3) establish the Nox-ROS-TRPM2 signaling pathway in the enhanced vasoreactivity and PASMC proliferation associated with PH. This proposal will provide novel information on the mechanism of pathogenesis in PH and identify potential targets for the development of new therapeutic strategies.

肺高压发病机制不明，无特效治疗，预后差。肺血管细胞ROS来源于NADPH氧化酶(Nox)催化，并参与多种病理生理过程，但ROS的Ca2+依赖性通路尚不明确。肺血管细胞表达TRPM2，有报道在非平滑肌细胞上TRPM2介导ROS(H2O2)诱导的Ca2+反应。本课题综合使用电生理、钙荧光成像、分子生物学、基因敲除和过表达以及生理学等技术，在正常大鼠、三种肺高压模型大鼠和 2种转基因小鼠上，试图明确：①哪种特异性Nox亚型(亚单位)参与何种肺高压发病过程。②ROS诱导Ca2+反应中，TRPM2激活机制，③Nox-ROS-TRPM2信号通路在与肺高压相关的血管高反应性和细胞增殖中的作用。为肺高压发病机制的阐明提供理论依据，并为其治疗提供新靶点。研究成果将丰富平滑肌细胞Ca2+信号调控理论，所揭示Nox/ROS/TRPM2信号通路作用，无论是对肺高压，或是其他心血管疾病发病机制的阐明都将提供新视点。

肺动脉平滑肌细胞（PASMCs）的活性氧（ROS）来源于NADPH氧化酶(Nox)催化，并参与多种病理生理过程。本研究试图阐明Nox-ROS-TRPM2通路在肺高压发病过程中激动剂引起血管高反应性和PASMCs增殖中的作用及机制。结果表明：.1）Nox1和Nox4亚型在CH致肺高压大鼠中表达量增加，Nox2亚型的表达在野百合碱（MCT）致肺高压大鼠中发生上调。.2）Nox1、Nox2和Nox4亚型均参与5-HT引起的收缩反应过程，Nox1亚型在该过程中起重要作用。而血管紧张素II （Ang II：Nox1和Nox2）或内皮素（ET-1：Nox1）引起收缩反应中只要部分Nox亚型发挥作用。.3）CH和MCT致肺高压中，无论是肺动脉主干（PAs）还是肺内动脉分枝（sPAs）对5-HT都呈现出高反应性，且sPAs对5-HT反应性高于PAs。.4）CH组，5-HT引起PAs高反应中Nox起重要作用（Nox1> Nox2> Nox4）；而MCT组，三种Nox亚型均部分参与MCT组中5-HT引起PAs高反应性。.5）在CON组5-HT可促进ROS产生，介导5-HT促进PASMCs产生ROS作用强度，Nox1/4 > Nox2 > Nox1，CH预处理促进PASMCs产生ROS作用较CON组显著增强。.6）两种肺高压，5-HT引起PAs高反应性是通过ROS-ADPR-TRPM2介导的，在CH组所发挥的可能作用大于MCT组。.7）CH组大鼠PASMCs具有较高水平基础Ca2+，5-HT引起PASMCs的Ca2+反应也增高，这种高Ca2+反应性可能是有由NOX1/4-TRPM2介导的。.8）5-HT可促进PASMCs增殖和迁移，Nox1/4参与5-HT和CH预处理促进PASMCs增殖和迁移。5-HT和CH预处理均可抑制PASMCs凋亡，在CON组Nox1/4亚型介导PASMCs的凋亡。.9）Nox4基因敲除小鼠可抵抗CH预处理3周所致肺高压的形成，进一步提示Nox4亚型在CH致肺高压中起重要作用。.10）CD38产物ADPR作为内源性的TRPM2激动剂，通过作用于多种嘌呤能2受体（P2X和P2Y）激活PASMCs的Ca2+反应，影响细胞增殖。.研究结果丰富平滑肌细胞Ca2+信号调控理论，所揭示Nox1/4/ROS/TRPM2信号通路作用，无论是对肺高压，或是其他心血管疾病发病机制的阐